Notch ligands regulate the muscle stem-like state ex vivo but are not sufficient for retaining regenerative capacity

Myogenic stem cells are a promising avenue for the treatment of muscular disorders. Freshly isolated muscle stem cells have a remarkable engraftment ability in vivo, but their cell number is limited. Current conventional culture conditions do not allow muscle stem cells to expand in vitro with their bona fide engraftment efficiency, requiring the improvement of culture procedures for achieving successful cell-therapy for muscle disorders. Here we expanded mouse muscle stem cells and human myoblasts with Notch ligands, DLL1, DLL4, and JAG1 to activate Notch signaling in vitro and to investigate whether these cells could retain their engraftment efficiency. Notch signaling promotes the expansion of Pax7+MyoD- mouse muscle stem-like cells and inhibits differentiation even after passage in vitro. Treatment with Notch ligands induced the Notch target genes and generated PAX7+MYOD- stem-like cells from human myoblasts previously cultured on conventional culture plates. However, cells treated with Notch ligands exhibit a stem cell-like state in culture, yet their regenerative ability was less than that of freshly isolated cells in vivo and was comparable to that of the control. These unexpected findings suggest that artificial maintenance of Notch signaling alone is insufficient for improving regenerative capacity of mouse and human donor-muscle cells and suggest that combinatorial events are critical to achieve muscle stem cell and myoblast engraftment potential.

肌源性干细胞（Myogenic stem cells）是治疗肌肉疾病的极具前景的途径。新鲜分离的肌肉干细胞在体内具备优异的植入能力，但其细胞数量十分有限。当前常规培养条件无法使肌肉干细胞在体外扩增的同时保留其真实的植入效率，因此亟需优化培养流程，以实现肌肉疾病的成功细胞治疗。本研究采用Notch配体（Notch ligands）DLL1、DLL4及JAG1，在体外激活Notch信号通路，对小鼠肌肉干细胞与人类成肌细胞进行扩增，并探究这些细胞能否保留其植入能力。结果显示，Notch信号通路可促进Pax7+MyoD-小鼠肌肉干细胞样细胞的扩增，且即便在体外传代后仍可抑制其分化。经Notch配体处理后，可诱导Notch靶基因的表达，并从常规培养板中预先培养的人类成肌细胞中诱导生成PAX7+MYOD-干细胞样细胞。然而，经Notch配体处理的细胞在培养中虽呈现干细胞样状态，但其再生能力却低于体内新鲜分离的细胞，仅与对照组相当。这些意外的研究结果表明，仅通过人工维持Notch信号通路，不足以提升小鼠及人类供体肌肉细胞的再生能力，同时提示联合调控事件对于实现肌肉干细胞与成肌细胞的植入潜能至关重要。